1. by Pratima Chaurasia, Dmitriy Berenzon, and Ronald Hoffman
Chromatin-modifying agents promote the ex vivo production of functional human erythroid progenitor cells
by Pratima Chaurasia, Dmitriy Berenzon, and Ronald Hoffman
Blood
Volume 117(17):
April 28, 2011
©2011 by American Society of Hematology

2. Effect of chromatin-modifying agents on ex vivo expansion of CB-EPCs.
Effect of chromatin-modifying agents on ex vivo expansion of CB-EPCs. (A) Effect of chromatin-modifying agents on ex vivo expansion of CB-CD34+: CD34+ cells were isolated from 5 individual CB units and treated with different HDACIs including SAHA (1μM), VPA(1mM), and TSA (1nM) and their effect on the number of CD34+ and CD34+CD90+cells following 7 days of culture was determined. The fold expansion of CD34+and CD34+CD90+ cell numbers was determined by dividing the total numbers of viable cells expressing the phenotype by the number of primary cells expressing the same phenotype multiplied by 100. A significant difference was observed between degree of expansion of CD34+cells (P = .01) and CD34+CD90+ cells (P = .02) obtained with VPA-containing cultures compared with cultures containing cytokines alone (n = 5). (B) Effect of HDACIs on the absolute number of BFU-E + CFU-Mix: CD34+ cells from 5 individual CB collections treated with cytokines and SAHA (1μM), VPA (1mM), or TSA (1nM) for 7days. The numbers of hematopoietic colonies were enumerated after 14 days. A greater number of BFU-E + CFU-Mix were generated in the presence of cytokines plus each of the HDACI (VPA, P = .002; SAHA, P = .04; and TSA, P = .08) compared with cultures containing cytokines alone. ■, BFU-E + CFU-Mix. (C) Effect of VPA on the fate of a single CD34+ cells: single primary CD34+cells and/or CD34+ isolated after 7 days of culture in the presence of cytokines alone and/or cytokines + VPA were deposited into 96-well plate in triplicate and supplemented with SCF, Epo, and IL-3. The number and types of HPC (BFU-E, CFU-Mix, and CFU-GM) were determined after 14 days of incubation (*P = .005; n = 3).
Pratima Chaurasia et al. Blood 2011;117:
©2011 by American Society of Hematology

3. Phenotypic and genetic analyses of hematopoietic cells after ex vivo cell culture.
Phenotypic and genetic analyses of hematopoietic cells after ex vivo cell culture. (A) Phenotypic analyses of VPA-treated CD34+ cells: CD34+ cells after 7 days of culture in the presence of cytokines alone or cytokines + VPA were analyzed by flow cytometrically. The expression of erythroid-specific markers (CD36, CD71, and GPA) and nonerythroid markers (CD14, CD15, and CD19) was assessed with appropriate lineage-specific monoclonal antibodies. For each mAb, the corresponding anti-isotype antibody was used in parallel to test the specificity of staining. Dot plots represent the percent expression of particular markers in day-7 cultures (n = 4, 1 of the 3 representative experiments is shown). (A) Cytokines alone; (B) cytokines + VPA. (C-D) Fold change in expression levels of genes associated with HSC/HPC and erythroid commitment: effects of VPA treatment on the relative transcript levels of genes (Dnmt1, Bmi1, Smad5, Ezh2, Eklf, EpoR, GATA1, GATA2, and Pu.1) was calculated by SYBR Green Q-PCR. Total RNA was extracted from primary CD34+ cells (day 0) and CD34+ cells isolated after 7 days of culture in the presence of cytokines with or without VPA treatment. GAPDH and tubulin was used as internal housekeeping genes. A relative mean normalized fold change in mRNA expression of VPA-treated CD34+ cells and CD34+ cells from cultures with cytokines alone were compared with the expression of these same genes in primary CB-CD34+ cells. Measurements were obtained in triplicate and a negative control (lacking the cDNA template) was included in each assay. , cytokines alone; , cytokines + VPA (n = 4).
Pratima Chaurasia et al. Blood 2011;117:
©2011 by American Society of Hematology

4. Histone acetylation status of ex vivo–generated CD34+ cells and promoters of erythroid-specific genes.
Histone acetylation status of ex vivo–generated CD34+ cells and promoters of erythroid-specific genes. (A) Analyses of histone acetylation. Representative flow cytometric analysis of the histone H3K9 acetylation status of primary CD34+cells, VPA-treated CD34+ cells, and CD34+ cells cultured in the presence of cytokines alone after 7 days of culture. Cells were stained with H3K9 antibody to assess the acetylation level of lysine 9 residue of histone H3 (n = 3). One of 3 representative experiments is shown. (B) Analyses of acetylated histone H3K9/14 and H3K27 on the promoters of erythroid lineage-specific genes, a stem cell gene (c-Kit), and a nonhematopoietic gene (MyoD) of cytokines alone and VPA-treated CD34+ cells as determined by ChIP assay. A reduction of H3K9/14 and H3K27 acetylation was observed with all of the promoters on the CD34+ cells isolated from cell culture with cytokines alone compared with cells cultured cytokines + VPA. ChIP efficiency, in terms of the percentage of input DNA recovered by immunoprecipitation, was determined by Q-PCR (primers were designed within −1-kb promoters of the erythroid lineage-related genes). A sample with no antibody (No Ab) was used as a background control. The histogram represents mean percentage of fold change relative to input chromatin and SE (n = 3). (C) Percent acetylation of H3K9/14 and H3K27 relative to histone H3 in CD34+ cells from cultures containing cytokines alone and cytokines + VPA. The percentage of acetylation of H3K9/14 and H3K27 relative to total histone H3 was determined on the erythroid lineage-specific promoters, a stem cell gene (c-Kit), and a non hematopoietic gene (MyoD) of CD34+ cells isolated from cultures performed in the presence of cytokines alone and cytokines + VPA cultures after 7 days of incubation. Histogram represents mean ± SE of ChIP Q- PCR (n = 3).
Pratima Chaurasia et al. Blood 2011;117:
©2011 by American Society of Hematology

5. Characterization of the transfusion product.
Characterization of the transfusion product. (A) Morphologic appearance of cells within the transfusion product. (i) Day-7 cells possessed an agranular cytoplasm. The nuclei of the cells had an open chromatin pattern and prominent nucleoli. (ii) Day-9 cells were smaller with compact and relatively smaller nuclei. (iii) Erythroid islands characterized by normoblasts surrounding a macrophage were observed in day-9 cultures. Giemsa-Wright staining (×1000 magnification). (B-C) Phenotypic analyses of the transfusion product: FACS analyses of the cells after days 7 and 9 of incubation was performed by immunostaining with various mAbs to lineage-specific markers including CD36, CD71, GPA, CD14, CD15, CD19, and chemokine receptor marker CXCR4. One of 4 representative experiments is shown.
Pratima Chaurasia et al. Blood 2011;117:
©2011 by American Society of Hematology

6. Functional behavior of ex vivo–generated TPs in NOD/SCID mice.
Functional behavior of ex vivo–generated TPs in NOD/SCID mice. (A) Analyses of human erythroid cells in peripheral blood of NOD/SCID mice receiving the TP. The mean ± SD of the percentage of human erythroid cells in the blood of NOD/SCID mice on serial days as described by double staining with LDS (nuclei) and human GPA. LDS+GPA+ represents human EPCs (dotted line) and LDS−GPA+ represents erythrocytes (solid line). Evidence of human EPCs and/or erythrocytes was not observed in control mice (black line indicates LDS−GPA+; and LDS+GPA+, gray line). Two to 3 mice were analyzed in each group (n = 2). (B) Representative histogram of human erythroid cells present on day 5 in the peripheral blood of NOD/SCID mouse. Similar results were obtained in an additional experiment. (C) Detection of human globins (β and γ) in the NOD/SCID mouse blood 15 days following the infusion of the transfusion product. Primers of mouse α-globin, human β and γ globins were mixed together and expression of globin mRNA was analyzed from cDNA prepared from mouse blood on day 15 by RT-PCR. Hu-β (212 bp), hu-γ (165 bp), RhD antigen (90 bp), and mouse-α (122 bp) gene products are shown on a 1.8% agarose gel. Lanes M1 and M2 contain DNA marker (a 100-bp ladder and 50-bp ladder); lane 1, blood from control NOD/SCID mouse; and lane 2, blood from NOD/SCID mouse infused with the transfusion product. One representative of 2 experiments is shown. (D) Human cell engraftment in the marrow of NOD/SCID mice. (i) Mouse bone marrow was harvested and analyzed on day 15 (NOD/SCID mice, n = 2) and (ii) in separate series of experiment NOD/SCIDγcnull mice day 30 (n = 3) after the infusion of the TP.
Pratima Chaurasia et al. Blood 2011;117:
©2011 by American Society of Hematology